Hydrogen exchange (HX) methods developed in this lab are being used to study allosteric process in hemoglobin. A survey of the entire hemoglobin HX curve has been completed and those exchanging protons which respond to the allosteric structure change have been measured. These amount to only 1/3 of the total peptide protons and occur in cohesive, 1st-order kinetic sets. Seven such sets have been found with 4 to 20 H in each set. On going from oxyHb to the deoxy form, all the H in a given set change their HX rate by factors ranging from 14 to 10 to the fourth power. The local unfolding model of HX requires just this behavior and shows how the allosteric free energy impinging on each set can be calculated from the observed HX rate change. To locate each set of allosterically responsive protons in the protein, hemoglobin samples with one set at a time labeled will be fragmented and the fragments bearing the tritium will be identified. These experiments are also expected to elucidate the protein fluctuational mechanism that underlies the HX process. To determine how each allosteric change interacts with the others and cooperates to carry structure-change energy through the protein, we will study the effect on each responsive segment of chemical modifications specifically placed at allosterically important sites. Similarly the developement of each of these changes will be studied as a function of increasing degree of liganding with CO. One particular responsive set appears to reflect the summed allosteric free energy in Hb. The reality of this correlation will be studied in a series of modified, allosterically-compromised samples by comparing the residual energy registered by this set with the true allosteric energy, measured by comparing oxy and deoxy Hb subunit dissociation. Other experiments, involving NMR and flash-photolysis techniques, will be done to study kinetic and equilibrium aspects of O2-penetration into heme and non-heme proteins.